This invention relates to reagents useful as inhibitors of T-cell leukemia virus (TLV) replication, and in particular, as inhibitors of human T-cell leukemia viruses 1 and 2 (HTLV-I and HTLV-II) replication.
Adult T-cell leukemia (ATL) was first described by workers in Japan. Takatsuki et al., 9 Jpn. J. Clin. Oncol. 312, 1979. Since then, the disease has been found in other areas of the Orient, the Caribbean basin, South America and central Africa. The epidemiology has shown a clear association with the presence of a retrovirus known as human T-cell leukemia virus type I (HTLV-I). HTLV-I infection of lymphocytes in vitro results in immortalization of the same cell type as tumor cells, and HTLV-I has proven to be oncogenic in animal model systems, e.g., the rabbit.
HTLV-I was first identified in the HUT 102 cell line (Gallo et al., 43 Cancer Res. 3892, 1983), which was established from a patient diagnosed with cutaneous T-cell lymphoma. Yoshida et al., 79 Proc. Nat. Acad. Sci. USA 2031, 1982, demonstrated that another cell line, known as MT-1, derived from a patient with ATL, also harbored a C-type retrovirus which was later named HTLV-I.
Since the initial description of ATL and discovery of HTLV-I, the virus has been shown to be associated with other human diseases. The most notable of these is a neurologic disorder known as tropical spastic parapesis (TSP), or HTLV-I associated myelopathy (HAM).
HTLV-II was first described in a T-cell line derived from a patient diagnosed with hairy-cell leukemia. The cell line (Mo-T) was derived from splenic tissue and later shown to harbor a virus which was related to (but distinct from) HTLV-I, and subsequently named HTLV-II. Like HTLV-I, HTLV-II will immortalize lymphocytes. However, limited numbers of HTLV-II associations with disease has precluded epidemiologic demonstration of an etiologic role of the virus in human malignancy.
HTLV is a member of the class of retroviruses that includes bovine leukemia virus (BLV) and simian T-cell leukemia virus (STLV-I). The viruses are grouped within the oncovirus subfamily and are distinct from the other human pathogenic retroviruses, e.g., human immunodeficiency virus (HIV), which belong to the subfamily of lentiviruses. Although the leukemia viruses are oncogenic, they do not harbor oncogenic sequences derived from cellular genomes and their replication is regulated by at least three other genes, known as tax, rex, and pro.
Three mRNA species have been identified for HTLV. The full length RNA, transcribed from the U3-R junction in the 5'LTR and terminating in the R-U5 region of the 3'LTR, is used for the synthesis of gag and pol gene products, and serves as the genomic RNA packaged into virions. A subgenomic RNA with one intron removed serves as the template for env protein synthesis, and a second subgenomic RNA with a second intron removed serves to encode the tax and rex products. The tax initiator codon is the same as that of the env protein and is encoded within the second exon. The rex initiator codon is also within the second exon but is located 59 nucleotides 5' to that of the tax and env.
Unlike the protease genes of the other retroviruses, the HTLV protease gene is encoded by a reading frame which spans the 3' part of the gag region and the 5' part of the pol region, but is distinct from both of the other ORFs. In some of the clones of HTLV-I, mutations in the protease region may be responsible for the lack of infectivity of the clones. The protease is responsible for processing mature gag products and autocatalyzed self-cleavage to produce the mature protease molecule.
Two unique genes are found in HTLV (tax and rex) which encode nonvirion proteins translated from a single, sliced mRNA with three exons. Deletion of sequences within these two genes render infectious clones of HTLV-II noninfectious, providing evidence that the expression of these genes is critical for viral replication.
The HTLV-I and HTLV-II tax genes encode proteins of 40 and 37 kd, respectively. The tax proteins are trans-acting transcriptional activators which increase the rate of transcription initiation from the provital 5'LTR promoter. Using cotransfection techniques, it has been shown that the tax protein is capable of trans-activating heterologous promoters, including the promoters for IL-2 and GM-CSF, c-fos and c-sis.
The rex gene of HTLV-I/HTLV-II encodes proteins of 27 and 21 kd for HTLV-I, and 26 and 24 kd for HTLV-II. The relationship between the two protein species is uncertain. Evidence suggests that the smaller rex protein is synthesized from an internal initiation codon within the same open reading frame. Both rex proteins are phosphorylated and localized in the nucleus of infected cells.
Like the product of the tax gene, the rex protein is essential to the replication of HTLV. Unlike the tax protein, the rex product appears to act post-transcriptionally to regulate viral gene expression. For HTLV-I, rex has been shown to increase the ratio of non-spliced RNA to the fully spliced mRNA which encodes tax and rex. In HTLV-II, rex protein increases the overall level of trans-activation in concert with tax protein. Additionally, in HTLV-II rex has a negative regulatory role which decreases viral mRNA levels. The ultimate effect of rex upon HTLV-I and HTLV-II infected cells is to regulate the levels of expression of genes encoding virion components, thereby determining the production of infectious virions.
The HTLV R and U5 regions are unusually long in comparison to other retroviruses. These regions encode the non-translated leader sequences at the 5' ends of all viral ImRNAs, exhibit extensive secondary structure and probably play a role in the control of translation of those mRNAs.
HTLV-I and HTLV-II share about 65% overall sequence homology at the nucleotide level. The homology is lowest in the LTR and non-translated regions and is highest in the tax and rex genes. Among different HTLV-I isolates, there is 96-99% homology in these two regions. Sequence homology is equally high among the HTLV-II isolates.
Direct cell-to-cell contact appears to be required for efficient HTLV infection. In vitro infection is usually accomplished by cocultivation of target cells with X-ray-irradiated or mitomycin C-treated virus producing cells. Infection by HTLV of susceptible cells is inefficient, with a slow course, as compared to other retrovirus infections. Infection of human cord or peripheral blood lymphocytes by HTLV results in virus production, and eventual immortalization of the cells. Only T-cells have been shown to be transformed by HTLV. Viral replication can occur in other cell types under certain circumstances, such as EBV-transformed B-cells. Productive infection has been observed in a few cells of nonlymphoid origin, most notably human endothelial cells and diploid human fibroblasts.
Infection of cells with HTLV in vitro can usually be accomplished only by cocultivation of the cells to be transformed with virus-infected cells. Proliferating transformed cells predominate in the cultures after 4 or more weeks. Transformed lymphocytes are mostly of the CD4.sup.+ phenotype which reflects the observation that tumors in ATL patients are almost invariably of CD4.sup.+ type. However, occasional CD8.sup.+ tumors have been reported and HTLV can transform CD8.sup.+ cells in vitro. Cells transformed in vitro transcribe RNA and produce virions, and these cells can be used to transform other cells. The lack of HTLV gene expression in ATL tumor cells suggests that in vitro transformation does not directly parallel the formation of a leukemic clone in vivo. These differences may reflect differences in selection pressure between in vitro and in vivo transformed clones.
Virion preparations from supernatants of HTLV-infected cells are mitogenic for quiescent human T-cells. Activated T-cells are more easily transformed by HTLV than are quiescent cells. This mitogenic activity of HTLV may be an important evolutionary adaptation because 95% of the T-cells in the body are generally in a quiescent state.
The mitogenic activity of virions suggests a mechanism for HTLV transformation whereby continual stimulation of T-cells via a T-cell receptor results in continuous cell proliferation. The corequisite of tax protein expression resembles the situation observed in transformation of primary rodent fibroblasts in vitro, where coexpression of two oncogenes, one acting in the nucleus and the other at the cell membrane, are required to effect transformation of the cell. Thus, unlike with other retroviruses, it appears that T-cell transformation is a normal course of events for infection with HTLV.
Several categories have been described as stages in ATL. These stages include: (i) asymptomatic carriers; (ii) preleukemic state (pre-ATL); (iii) chronic/smoldering ATL; and (iv) acute ATL. In some cases, these stages may be temporally related. The only significant prognostic factor in acute ATL is the presence of ascites, which is associated with shorter-survival. Despite aggressive chemotherapy, the median survival in acute ATL is measured in months.
The form of disease in some ATL patients is that of a T-cell lymphoma rather than a leukemia. The differential diagnosis of ATL includes other T-cell malignancies such as non-Hodgkins lymphoma, mycosis fungoides, Sezary's syndrome and T-cell chronic lymphocytic leukemia (CLL). One malignancy distinct from ATL in which HTLV-I may play a role is B-cell chronic lymphocytic leukemia (B-cell CLL). There is some evidence for functional impairment of the immune response in ATL patients, as well as some HTLV-I carriers. The role of HTLV-II in the few cases of leukemia associated with viral infection is unclear, since insufficient cases are available for study.
ATL is a highly malignant disease where survival of subacute or acute disease is measured in months. Since only about 1% of patients progress from asymptomatic to acute disease, treatment is reserved for more advanced disease. Standard combination therapies which are used for other aggressive lymphomas and leukemias have not worked for ATL. Other agents such as deoxycoformycin, beta interferon, gamma interferon and anti-Tac antiboby have not been of value in treating ATL.